Protein activity screening of clones having dna from uncultivated microorganisms

ABSTRACT

Disclosed is a process of screening clones having DNA from an uncultivated microorganism for a specified protein, e.g. enzyme, activity by screening for a specified protein, e.g. enzyme, activity in a library of clones prepared by (i) recovering DNA from a DNA population derived from at least one uncultivated microorganism; and (ii) transforming a host with recovered DNA to produce a library of clones which is screened for the specified protein, e.g. enzyme, activity.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 08/568,994 which was filed on Dec. 7, 1995 (copending) which isa continuation-in-part of U.S. application Ser. No. 08/503,606 which wasfiled on Jul. 18, 1995 (copending).

[0002] This invention relates to the field of preparing and screeninglibraries of clones containing microbially derived DNA.

[0003] Naturally occurring assemblages of microorganisms often encompassa bewildering array of physiological and metabolic diversity. In fact,it has been estimated that to date less than one percent of the world'sorganisms have been cultured. It has been suggested that a largefraction of this diversity thus far has been unrecognized due todifficulties in enriching and isolating microorganisms in pure culture.Therefore, it has been difficult or impossible to identify or isolatevaluable proteins, e.g. enzymes, from these samples. These limitationssuggest the need for alternative approaches to characterize thephysiological and metabolic potential, i.e. activities of interest ofas-yet uncultivated microorganisms, which to date have beencharacterized solely by analyses of PCR amplified rRNA gene fragments,clonally recovered from mixed assemblage nucleic acids.

[0004] In one aspect, the invention provides a process of screeningclones having DNA from an uncultivated microorganism for a specifiedprotein, e.g. enzyme, activity which process comprises:

[0005] screening for a specified protein, e.g. enzyme, activity in alibrary of clones prepared by

[0006] (i) recovering DNA from a DNA population derived from at leastone uncultivated microorganism; and

[0007] (ii) transforming a host with recovered DNA to produce a libraryof clones which are screened for the specified protein, e.g. enzyme,activity.

[0008] The library is produced from DNA which is recovered withoutculturing of an organism, particularly where the DNA is recovered froman environmental sample containing microorganisms which are not orcannot be cultured.

[0009] In a preferred embodiment DNA is ligated into a vector,particularly wherein the vector further comprises expression regulatorysequences which can control and regulate the production of a detectableproteins, e.g. enzyme, activity from the ligated DNA.

[0010] The f-factor (or fertility factor) in E. coli is a plasmid whicheffects high frequency transfer of itself during conjugation and lessfrequent transfer of the bacterial chromosome itself. To achieve andstably propogate large DNA fragments from mixed microbial samples, aparticularly preferred embodiment is to use a cloning vector containingan f-factor origin of replication to generate genomic libraries that canbe replicated with a high degree of fidelity. When integrated with DNAfrom a mixed uncultured environmental sample, this makes it possible toachieve large genomic fragments in the form of a stable “environmentalDNA library.”

[0011] In another preferred embodiment, double stranded DNA obtainedfrom the uncultivated DNA population is selected by:

[0012] converting the double stranded genomic DNA into single strandedDNA;

[0013] recovering from the converted single stranded DNA single strandedDNA which specifically binds, such as by hybridization, to a probe DNAsequence; and

[0014] converting recovered single stranded DNA to double stranded DNA.

[0015] The probe may be directly or indirectly bound to a solid phase bywhich it is separated from single stranded DNA which is not hybridizedor otherwise specifically bound to the probe.

[0016] The process can also include releasing single stranded DNA fromsaid probe after recovering said hybridized or otherwise bound singlestranded DNA and amplifying the single stranded DNA so released prior toconverting it to double stranded DNA.

[0017] The invention also provides a process of screening clones havingDNA from an uncultivated microorganisms for a specified protein, e.g.enzyme, activity which comprises screening for a specified gene clusterprotein product activity in the library of clones prepared by: (i)recovering DNA from a DNA population derived from at least oneuncultivated microorganism; and (ii) transforming a host with recoveredDNA to produce a library of clones with the screens for the specifiedprotein, e.g. enzyme, activity. The library is produced from genecluster DNA which is recovered without culturing of an organism,particularly where the DNA gene clusters are recovered from anenvironmental sample containing microorganisms which are not or cannotbe cultured.

[0018] Alternatively, double-stranded gene cluster DNA obtained from theuncultivated DNA population is selected by converting thedouble-stranded genomic gene cluster DNA into single-stranded DNA;recovering from the converted single-stranded gene cluster polycistronDNA, single-stranded DNA which specifically binds, such as byhybridization, to a polynucleotide probe sequence; and convertingrecovered single-stranded gene cluster DNA to double-stranded DNA.

[0019] These and other aspects of the present invention are describedwith respect to particular preferred embodiments and will be apparent tothose skilled in the art from the teachings herein.

[0020] The microorganisms from which the libraries may be preparedinclude prokaryotic microorganisms, such as Eubacteria andArchaebacteria, and lower eukaryotic microorganisms such as fungi, somealgae and protozoa. The microorganisms are uncultured microorganismsobtained from environmental samples and such microorganisms may beextremophiles, such as thermophiles, hyperthermophiles, psychrophiles,psychrotrophs, etc.

[0021] As indicated above, the library is produced from DNA which isrecovered without culturing of an organism, particularly where the DNAis recovered from an environmental sample containing microorganismswhich are not or cannot be cultured. Sources of microorganism DNA as astarting material library from which DNA is obtained are particularlycontemplated to include environmental samples, such as microbial samplesobtained from Arctic and Antarctic ice, water or permafrost sources,materials of volcanic origin, materials from soil or plant sources intropical areas, etc. Thus, for example, genomic DNA may be recoveredfrom either uncultured or non-culturable organism and employed toproduce an appropriate library of clones for subsequent determination ofprotein, e.g. enzyme, activity.

[0022] Bacteria and many eukaryotes have a coordinated mechanism forregulating genes whose products are involved in related processes. Thegenes are clustered, in structures referred to as “gene clusters,” on asingle chromosome and are transcribed together under the control of asingle regulatory sequence, including a single promoter which initiatestranscription of the entire cluster. The gene cluster, the promoter, andadditional sequences that function in regulation altogether are referredto as an “operon” and can include up to 20 or more genes, usually from 2to 6 genes. Thus, a gene cluster is a group of adjacent genes that areeither identical or related, usually as to their function.

[0023] Some gene families consist of identical members. Clustering is aprerequisite for maintaining identity between genes, although clusteredgenes are not necessarily identical. Gene clusters range from extremeswhere a duplication is generated to adjacent related genes to caseswhere hundreds of identical genes lie in a tandem array. Sometimes nosignificance is discernable in a repetition of a particular gene. Aprincipal example of this is the expressed duplicate insulin genes insome species, whereas a single insulin gene is adequate in othermammalian species.

[0024] It is important to further research gene clusters and the extentto which the full length of the cluster is necessary for the expressionof the proteins resulting therefrom. Further, gene clusters undergocontinual reorganization and, thus, the ability to create heterogeneouslibraries of gene clusters from, for example, bacterial or otherprokaryote sources is valuable in determining sources of novel proteins,particularly including proteins, e.g. enzymes, such as, for example, thepolyketide synthases that are responsible for the synthesis ofpolyketides having a vast array of useful activities. Other types ofproteins that are the product(s) of gene clusters are also contemplated,including, for example, antibiotics, antivirals, antitumor agents andregulatory proteins, such as insulin.

[0025] Polyketides are molecules which are an extremely rich source ofbioactivities, including antibiotics (such as tetracyclines anderythromycin), anti-cancer agents (daunomycin), immunosuppressants(FK506 and rapamycin), and veterinary products (monensin). Manypolyketides (produced by polyketide synthases) are valuable astherapeutic agents. Polyketide synthases are multifunctional proteins,e.g. enzymes, that catalyze the biosynthesis of a hugh variety of carbonchains differing in length and patterns of functionality andcyclization. Polyketide synthase genes fall into gene clusters and atleast one type (designated type I) of polyketide synthases have largesize genes and proteins, e.g. enzymes, complicating genetic manipulationand in vitro studies of these genes/proteins.

[0026] The ability to select and combine desired components from alibrary of polyketides and postpolyketide biosynthesis genes forgeneration of novel polyketides for study is appealing. The method(s) ofthe present invention make it possible to and facilitate the cloning ofnovel polyketide synthases, since one can generate gene banks withclones containing large inserts (especially when using the f-factorbased vectors), which facilitates cloning of gene clusters.

[0027] Preferably, the gene cluster DNA is ligated into a vector,particularly wherein a vector further comprises expression regulatorysequences which can control and regulate the production of a detectableprotein or protein-related array activity from the ligated geneclusters. Use of vectors which have an exceptionally large capacity forexogenous DNA introduction are particularly appropriate for use withsuch gene clusters and are described by way of example herein to includethe f-factor (or fertility factor) of E. coli. This f-factor of E. coliis a plasmid which affect high-frequency transfer of itself duringconjugation and is ideal to achieve and stably propagate large DNAfragments, such as gene clusters from mixed microbial samples.

[0028] The term “derived” or “isolated” means that material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide separated from some or all of thecoexisting materials in the natural system, is isolated.

[0029] The DNA isolated or derived from these microorganisms canpreferably be inserted into a vector prior to probing for selected DNA.Such vectors are preferably those containing expression regulatorysequences, including promoters, enhancers and the like. Suchpolynucleotides can be part of a vector and/or a composition and stillbe isolated, in that such vector or composition is not part of itsnatural environment. Particularly preferred phage or plasmid and methodsfor introduction and packaging into them are described in detail in theprotocol set forth herein.

[0030] The following outlines a general procedure for producinglibraries from non-culturable organisms, which libraries can be probedto select therefrom DNA sequences which hybridize to specified probeDNA:

[0031] Obtain Biomass

[0032] DNA Isolation

[0033] Shear DNA (25 gauge needle)

[0034] Blunt DNA (Mung Bean Nuclease)

[0035] Methylate (EcoR I Methylase)

[0036] Ligate to EcoR I linkers (GGAATTCC)

[0037] Cut back linkers (EcoR I Restriction Endonuclease)

[0038] Size Fractionate (Sucrose Gradient)

[0039] Ligate to lambda vector (Lambda ZAP II and gt11)

[0040] Package (in vitro lambda packaging extract)

[0041] Plate on E. coli host and amplify

[0042] The probe DNA used for selectively recovering DNA of interestfrom the DNA derived from the at least one uncultured microorganism canbe a full-length coding region sequence or a partial coding regionsequence of DNA for an protein, e.g. enzyme, of known activity, aphylogenetic marker or other identified DNA sequence. The original DNAlibrary can be preferably probed using mixtures of probes comprising atleast a portion of the DNA sequence encoding the specified activity.These probes or probe libraries are preferably single-stranded and themicrobial DNA which is probed has preferably been converted intosingle-stranded form. The probes that are particularly suitable arethose derived from DNA encoding proteins, e.g. enzymes, having anactivity similar or identical to the specified protein, e.g. enzyme,activity which is to be screened.

[0043] The probe DNA should be at least about 10 bases and preferably atleast 15 bases. In one embodiment, the entire coding region may beemployed as a probe. Conditions for the hybridization in which DNA isselectively isolated by the use of at least one DNA probe will bedesigned to provide a hybridization stringency of at least about 50%sequence identity, more particularly a stringency providing for asequence identity of at least about 70%.

[0044] Hybridization techniques for probing a microbial DNA library toisolate DNA of potential interest are well known in the art and any ofthose which are described in the literature are suitable for use herein,particularly those which use a solid phase-bound, directly or indirectlybound, probe DNA for ease in separation from the remainder of the DNAderived from the microorganisms.

[0045] Preferably the probe DNA is “labeled” with one partner of aspecific binding pair (i.e. a ligand) and the other partner of the pairis bound to a solid matrix to provide ease of separation of target fromits source. The ligand and specific binding partner can be selectedfrom, in either orientation, the following: (1) an antigen or hapten andan antibody or specific binding fragment thereof; (2) biotin oriminobiotin and avidin or streptavidin; (3) a sugar and a lectinspecific therefor; (4) a protein, e.g. enzyme, and an inhibitortherefor; (5) an apoenzyme and cofactor; (6) complementary homopolymericoligonucleotides; and (7) a hormone and a receptor therefor. The solidphase is preferably selected from: (1) a glass or polymeric surface; (2)a packed column of polymeric beads; and (3) magnetic or paramagneticparticles.

[0046] The library of clones prepared as described above can be screeneddirectly for enzymatic activity without the need for culture expansion,amplification or other supplementary procedures. However, in onepreferred embodiment, it is considered desirable to amplify the DNArecovered from the individual clones such as by PCR.

[0047] Further, it is optional but desirable to perform an amplificationof the target DNA that has been isolated. In this embodiment theselectively isolated DNA is separated from the probe DNA afterisolation. It is then amplified before being used to transform hosts.The double stranded DNA selected to include as at least a portionthereof a predetermined DNA sequence can be rendered single stranded,subjected to amplification and reannealed to provide amplified numbersof selected double stranded DNA. Numerous amplification methodologiesare now well known in the art.

[0048] The selected DNA is then used for preparing a library forscreening by transforming a suitable organism. Hosts, particularly thosespecifically identified herein as preferred, are transformed byartificial introduction of the vectors containing the target DNA byinoculation under conditions conducive for such transformation.

[0049] The resultant libraries of transformed clones are then screenedfor clones which display activity for the protein, e.g. enzyme, ofinterest in a phenotypic assay for protein, e.g. enzyme, activity.

[0050] Having prepared a multiplicity of clones from DNA selectivelyisolated from an organism, such clones are screened for a specificprotein, e.g. enzyme, activity and to identify the clones having thespecified protein, e.g. enzyme, characteristics.

[0051] The screening for protein, e.g. enzyme, activity may be effectedon individual expression clones or may be initially effected on amixture of expression clones to ascertain whether or not the mixture hasone or more specified protein, e.g. enzyme, activities. If the mixturehas a specified protein, e.g. enzyme, activity, then the individualclones may be rescreened for such protein, e.g. enzyme, activity or fora more specific activity. Thus, for example, if a clone mixture hashydrolase activity, then the individual clones may be recovered andscreened to determine which of such clones has hydrolase activity.

[0052] The DNA derived from a microorganism(s) is preferably insertedinto an appropriate vector (generally a vector containing suitableregulatory sequences for effecting expression) prior to subjecting suchDNA to a selection procedure to select and isolate therefrom DNA whichhybridizes to DNA derived from DNA encoding an proteins, e.g. enzyme(s),having the specified protein, e.g. enzyme, activity.

[0053] As representative examples of expression vectors which may beused there may be mentioned viral particles, baculovirus, phage,plasmids, phagemids, cosmids, phosmids, bacterial artificialchromosomes, viral DNA (e.g. vaccinia, adenovirus, foul pox virus,pseudorabies and derivatives of SV40), P1-based artificial chromosomes,yeast plasmids, yeast artificial chromosomes, and any other vectorsspecific for specific hosts of interest (such as bacillus, aspergillus,yeast, etc.) Thus, for example, the DNA may be included in any one of avariety of expression vectors for expressing a polypeptide. Such vectorsinclude chromosomal, nonchromosomal and synthetic DNA sequences. Largenumbers of suitable vectors are known to those of skill in the art, andare commercially available. The following vectors are provided by way ofexample; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBluescript SK,pBluescript KS (Stratagene); pTRC99a, pKK223-3, pDR540, pRIT2T(Pharmacia); Eukaryotic: pWLNEO, pXT1, pSG5 (Stratagene) pSVK3, pBPV,pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or vector may beused as long as they are replicable and viable in the host.

[0054] A particularly preferred type of vector for use in the presentinvention contains an f-factor origin of replication. The f-factor (orfertility factor) in E. coli is a plasmid which effects high frequencytransfer of itself during conjugation and less frequent transfer of thebacterial chromosome itself. A particularly preferred embodiment is touse cloning vectors, referred to as “fosmids” or bacterial artificialchromosome (BAC) vectors. These are derived from the E. coli f-factorand are able to stably integrate large segments of genomic DNA. Whenintegrated with DNA from a mixed uncultured environmental sample, thismakes it possible to achieve large genomic fragments in the form of astable “environmental DNA library.”

[0055] The DNA derived from a microorganism(s) may be inserted into thevector by a variety of procedures. In general, the DNA sequence isinserted into an appropriate restriction endonuclease site(s) byprocedures known in the art. Such procedures and others are deemed to bewithin the scope of those skilled in the art.

[0056] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. Particular named bacterial promoters include lacI, lacZ,T3, T7, gpt, lambda P_(R), P_(L) and trp. Eukaryotic promoters includeCMV immediate early, HSV thymidine kinase, early and late SV40. LTRsfrom retrovirus, and mouse metallothionein-I. Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art. The expression vector also contains a ribosome bindingsite for translation initiation and a transcription terminator. Thevector may also include appropriate sequences for amplifying expression.Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers.

[0057] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0058] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic proteins, e.g. enzymes, such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium.

[0059] The DNA selected and isolated as hereinabove described isintroduced into a suitable host to prepare a library which is screenedfor the desired protein, e.g. enzyme, activity. The selected DNA ispreferably already in a vector which includes appropriate controlsequences whereby selected DNA which encodes for an protein, e.g.enzyme, may be expressed, for detection of the desired activity. Thehost cell can be a higher eukaryotic cell, such as a mammalian cell, ora lower eukaryotic cell, such as a yeast cell, or the host cell can be aprokaryotic cell, such as a bacterial cell. Introduction of theconstruct into the host cell can be effected by transformation, calciumphosphate transfection, DEAE-Dextran mediated transfection, DMSO orelectroporation (Davis, L., Dibner, M., Battey, I., Basic Methods inMolecular Biology, (1986)).

[0060] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Bacillus, Streptomyces,Salmonella typhimurium; fungal cells, such as yeast; insect cells suchas Drosophila S2 and Spodoptera Sƒ9; animal cells such as CHO, COS orBowes melanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0061] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors. The engineered host cells can becultured in conventional nutrient media modified as appropriate foractivating promoters, selecting transformants or amplifying genes. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0062] The library may be screened for a specified protein, e.g. enzyme,activity by procedures known in the art. For example, the protein, e.g.enzyme, activity may be screened for one or more of the six IUB classes;oxidoreductases, transferases, hydrolases, lyases, isomerases andligases. The recombinant proteins, e.g. enzymes, which are determined tobe positive for one or more of the IUB classes may then be rescreenedfor a more specific protein, e.g. enzyme, activity.

[0063] Alternatively, the library may be screened for a more specializedprotein, e.g. enzyme, activity. For example, instead of genericallyscreening for hydrolase activity, the library may be screened for a morespecialized activity, i.e. the type of bond on which the hydrolase acts.Thus, for example, the library may be screened to ascertain thosehydrolases which act on one or more specified chemical functionalities,such as: (a) amide (peptide bonds), i.e. proteases; (b) ester bonds,i.e. esterases and lipases; (c) acetals, i.e., glycosidases etc.

[0064] The clones which are identified as having the specified protein,e.g. enzyme, activity may then be sequenced to identify the DNA sequenceencoding an protein, e.g. enzyme, having the specified activity. Thus,in accordance with the present invention it is possible to isolate andidentify: (i) DNA encoding an protein, e.g. enzyme, having a specifiedprotein, e.g. enzyme, activity, (ii) proteins, e.g. enzymes, having suchactivity (inlcuding the amino acid sequence thereof) and (iii) producerecombinant proteins, e.g. enzymes, having such activity.

[0065] The present invention may be employed for example, to identifyuncultured microorganisms with proteins, e.g. enzymes, having, forexample, the following activities which may be employed for thefollowing uses:

[0066] 1 Lipase/Esterase

[0067] a. Enantioselective hydrolysis of esters (lipids)/thioesters

[0068] 1) Resolution of racemic mixtures

[0069] 2) Synthesis of optically active acids or alcohols frommeso-diesters

[0070] b. Selective syntheses

[0071] 1) Regiospecific hydrolysis of carbohydrate esters

[0072] 2) Selective hydrolysis of cyclic secondary alcohols

[0073] c. Synthesis of optically active esters, lactones, acids,alcohols

[0074] 1) Transesterification of activated/nonactivated esters

[0075] 2) Interesterification

[0076] 3) Optically active lactones from hydroxyesters

[0077] 4) Regio- and enantioselective ring opening of anhydrides

[0078] d. Detergents

[0079] e. Fat/Oil conversion

[0080] f. Cheese ripening

[0081] 2 Protease

[0082] a. Ester/amide synthesis

[0083] b. Peptide synthesis

[0084] c. Resolution of racemic mixtures of amino acid esters

[0085] d. Synthesis of non-natural amino acids

[0086] e. Detergents/protein hydrolysis

[0087] 3 Glycosidase/Glycosyl transferase

[0088] a. Sugar/polymer synthesis

[0089] b. Cleavage of glycosidic linkages to form mono, di-andoligosaccharides

[0090] c. Synthesis of complex oligosaccharides

[0091] d. Glycoside synthesis using UDP-galactosyl transferase

[0092] e. Transglycosylation of disaccharides, glycosyl fluorides, arylgalactosides

[0093] f. Glycosyl transfer in oligosaccharide synthesis

[0094] g. Diastereoselective cleavage of β-glucosylsulfoxides

[0095] h. Asymmetric glycosylations

[0096] i. Food processing

[0097] j. Paper processing

[0098] 4 Phosphatase/Kinase

[0099] a. Synthesis/hydrolysis of phosphate esters

[0100] 1) Regio-, enantioselective phosphorylation

[0101] 2) Introduction of phosphate esters

[0102] 3) Synthesize phospholipid precursors

[0103] 4) Controlled polynucleotide synthesis

[0104] b. Activate biological molecule

[0105] c. Selective phosphate bond formation without protecting groups

[0106] 5 Mono/Dioxygenase

[0107] a. Direct oxyfunctionalization of unactivated organic substrates

[0108] b. Hydroxylation of alkane, aromatics, steroids

[0109] c. Epoxidation of alkenes

[0110] d. Enantioselective sulphoxidation

[0111] e. Regio- and stereoselective Bayer-Villiger oxidations

[0112] 6 Haloperoxidase

[0113] a. Oxidative addition of halide ion to nucleophilic sites

[0114] b. Addition of hypohalous acids to olefinic bonds

[0115] c. Ring cleavage of cyclopropanes

[0116] d. Activated aromatic substrates converted to ortho and paraderivatives

[0117] e. 1.3 diketones converted to 2-halo-derivatives

[0118] f. Heteroatom oxidation of sulfur and nitrogen containingsubstrates

[0119] g. Oxidation of enol acetates, alkynes and activated aromaticrings

[0120] 7 Lignin peroxidase/Diarylpropane peroxidase

[0121] a. Oxidative cleavage of C—C bonds

[0122] b. Oxidation of benzylic alcohols to aldehydes

[0123] c. Hydroxylation of benzylic carbons

[0124] d. Phenol dimerization

[0125] e. Hydroxylation of double bonds to form diols

[0126] f. Cleavage of lignin aldehydes

[0127] 8 Epoxide hydrolase

[0128] a. Synthesis of enantiomerically pure bioactive compounds

[0129] b. Regio- and enantioselective hydrolysis of epoxide

[0130] c. Aromatic and olefinic epoxidation by monooxygenases to formepoxides

[0131] d. Resolution of racemic epoxides

[0132] e. Hydrolysis of steroid epoxides

[0133] 9 Nitrile hydratase/nitrilase

[0134] a. Hydrolysis of aliphatic nitriles to carboxamides

[0135] b. Hydrolysis of aromatic, heterocyclic, unsaturated aliphaticnitriles to corresponding acids

[0136] c. Hydrolysis of acrylonitrile

[0137] d. Production of aromatic and carboxamides, carboxylic acids(nicotinamide, picolinamide, isonicotinamide)

[0138] e. Regioselective hydrolysis of acrylic dinitrile

[0139] f. α-amino acids from α-hydroxynitriles

[0140] 10 Transaminase

[0141] a. Transfer of amino groups into oxo-acids

[0142] 11 Amidase/Acylase

[0143] a. Hydrolysis of amides, amidines, and other C-N bonds

[0144] b. Non-natural amino acid resolution and synthesis

EXAMPLE 1 Preparation of a Mammalian DNA Library

[0145] The following outlines the procedures used to generate a genelibrary from a sample of the exterior surface of a whale bone found at1240 meters depth in the Santa Catalina Basin during a dive expedition.

[0146] Isolate DNA.

[0147] IsoQuick Procedure as per manufacturer's instructions.

[0148] Shear DNA

[0149] 1. Vigorously push and pull DNA through a 25G double-hub needleand 1-cc syringes about 500 times.

[0150] 2. Check a small amount (0.5 μg) on a 0.8% agarose gel to makesure the majority of the DNA is in the desired size range (about 3-6kb).

[0151] Blunt DNA

[0152] 1. Add: H₂O to a final volume of 405 μl  45 μl 10X Mung BeanBuffer 2.0 μl Mung Bean Nuclease (150 u/μl)

[0153] 2. Incubate 37° C., 15 minutes.

[0154] 3. Phenol/chloroform extract once.

[0155] 4. Chloroform extract once.

[0156] 5. Add 1 ml ice cold ethanol to precipitate.

[0157] 6. Place on ice for 10 minutes.

[0158] 7. Spin in microfuge, high speed, 30 minutes.

[0159] 8. Wash with 1 ml 70% ethanol.

[0160] 9. Spin in microfuge, high speed, 10 minutes and dry.

[0161] Methylate DNA

[0162] 1. Gently resuspend DNA in 26 μl TE.

[0163] 2. Add: 4.0 μl 10X EcoR I Methylase Buffer 0.5 μl SAM (32 mM) 5.0μl EcoR I Methylase (40 u/μl)

[0164] 3. Incubate 37°, 1 hour.

[0165] Insure Blunt Ends

[0166] 1. Add to the methylation reaction: 5.0 μl 100 mM MgCl₂ 8.0 μldNTP mix (2.5 mM of each dGTP, dATP, dTTP, dCTP) 4.0 μl Klenow (5 u/μl)

[0167] 2. Incubate 12° C., 30 minutes.

[0168] 3. Add 450 μl 1X STE.

[0169] 4. Phenol/chloroform extract once.

[0170] 5. Chloroform extract once.

[0171] 6. Add 1 ml ice cold ethanol to precipitate and place on ice for10 minutes.

[0172] 7. Spin in microfuge, high speed, 30 minutes.

[0173] 8. Wash with 1 ml 70% ethanol.

[0174] 9. Spin in microfuge, high speed, 10 minutes and dry.

[0175] Linker Ligation

[0176] 1. Gently resuspend DNA in 7 μl Tris-EDTA (TE).

[0177] 2. Add:  14 μl Phosphorylated EcoR I linkers (200 ng/μl) 3.0 μl10X Ligation Buffer 3.0 μl 10 mM rATP 3.0 μl T4 DNA Ligase (4 Wu/μl)

[0178] 3. Incubate 4° C., overnight.

[0179] EcoR1 Cutback

[0180] 1. Heat kill ligation reaction 68° C., 10 minutes.

[0181] 2. Add: 237.9 μl H₂O 30 μl 10X EcoR I Buffer 2.1 μl EcoR IRestriction Enzyme (100 u/μl)

[0182] 3. Incubate 37° C., 1.5 hours.

[0183] 4. Add 1.5 μl 0.5 M EDTA.

[0184] 5. Place on ice.

[0185] Sucrose Gradient (2.2 ml) Size Fractionation

[0186] 1. Heat sample to 65° C., 10 minutes.

[0187] 2. Gently load on 2.2 ml sucrose gradient.

[0188] 3. Spin in mini-ultracentrifuge, 45K., 20° C., 4 hours (nobrake).

[0189] 4. Collect fractions by puncturing the bottom of the gradienttube with a 20G needle and allowing the sucrose to flow through theneedle. Collect the first 20 drops in a Falcon 2059 tube then collect 101-drop fractions (labelled 1-10). Each drop is about 60 μl in volume.

[0190] 5. Run 5 μl of each fraction on a 0.8% agarose gel to check thesize.

[0191] 6. Pool fractions 1-4 (−10-1.5 kb) and, in a separate tube, poolfractions 5-7 (about 5-0.5 kb).

[0192] 7. Add 1 ml ice cold ethanol to precipitate and place on ice for10 minutes.

[0193] 8. Spin in microfuge, high speed, 30 minutes.

[0194] 9. Wash with 1 ml 70% ethanol.

[0195] 10. Spin in microfuge, high speed, 10 minutes and dry.

[0196] 11. Resuspend each in 10 μl TE buffer.

[0197] Test Ligation to Lambda Arms

[0198] 1. Plate assay to get an approximate concentration. Spot 0.5 μlof the sample on agarose containing ethidium bromide along withstandards (DNA samples of known concentration). View in UV light andestimate concentration compared to the standards. Fraction 1-4=>1.0μg/μl. Fraction 5-7=500 ng/μl.

[0199] 2. Prepare the following ligation reactions (5 μl reactions) andincubate 4° C., overnight: Lambda 10X arms T4 DNA Ligase 10 mM (gt11 andInsert Ligase (4 Sample H₂O Buffer rATP ZAP) DNA Wu/μ) Fraction 1-4 0.5μl 0.5 μl 0.5 μl 1.0 μl 2.0 μl 0.5 μl Fraction 5-7 0.5 μl 0.5 μl 0.5 μl1.0 μl 2.0 μl 0.5 μl

[0200] Test Package and Plate

[0201] 1. Package the ligation reactions following manufacturer'sprotocol. Package 2.5 μl per packaging extract (2 extracts perligation).

[0202] 2. Stop packaging reactions with 500 μl SM buffer and poolpackaging that came from the same ligation.

[0203] 3. Titer 1.0 μl of each on appropriate host (OD₆₀₀=1.0) [XLI-BlueMRF for ZAP and Y1088 for gt11]

[0204] Add 200 μl host (in mM MgSO₄) to Falcon 2059 tubes

[0205] Inoculate with 1 μl packaged phage

[0206] Incubate 37° C., 15 minutes

[0207] Add about 3 ml 48° C. top agar

[0208] [50 ml stock containing 150 μl IPTG (0.5M) and 300 μl X-GAL (350mg/ml))

[0209] Plate on 100 mm plates and incubate 37° C., overnight.

[0210] 4. Efficiency results:

[0211] gt11: 1.7×10⁴ recombinants with 95% background

[0212] ZAP II: 4.2×10⁴ recombinants with 66% background

[0213] Contaminants in the DNA sample may have inhibited the enzymaticreactions, though the sucrose gradient and organic extractions may haveremoved them. Since the DNA sample was precious, an effort was made to“fix” the ends for cloning:

[0214] Re-Blunt DNA

[0215] 1. Pool all left over DNA that was not ligated to the lambda arms(Fractions 1-7) and add H₂O to a final volume of 12 μl. Then add: 143 μlH₂O 20 μl 10X Buffer 2 (from Stratagene's cDNA Synthesis Kit) 23 μlBlunting dNTP (from Stratagene's cDNA Synthesis Kit) 2.0 μl Pfu (fromStratagene's cDNA Synthesis

[0216] 2. Incubate 72° C., 30 minutes.

[0217] 3. Phenol/chloroform extract once.

[0218] 4. Chloroform extract once.

[0219] 5. Add 20 μL 3M NaOAc and 400 μl ice cold ethanol to precipitate.

[0220] 6. Place at −20° C., overnight.

[0221] 7. Spin in microfuge, high speed,30 minutes.

[0222] 8. Wash with 1 ml 70% ethanol.

[0223] 9. Spin in microfuge, high speed, 10 minutes and dry.

[0224] (Do NOT Methylate DNA since it was already methylated in thefirst round of processing)

[0225] Adaptor Ligation

[0226] 1. Gently resuspend DNA in 8 μl EcoR I adaptors (fromStratagene's cDNA Synthesis Kit).

[0227] 2. Add: 1.0 μl 10X Ligation Buffer 1.0 μl 10 mM rATP 1.0 μl T4DNA Ligase (4 Wu/μl)

[0228] 3. Incubate 4° C., 2 days.

[0229] (Do NOT cutback since using ADAPTORS this time. Instead, need tophosphorylate)

[0230] Phosphorylate Adaptors

[0231] 1. Heat kill ligation reaction 70° C., 30 minutes.

[0232] Add: 1.0 μl 10X Ligation Buffer 2.0 μl 10 mM rATF 6.0 μl H₂O 1.0μl PNK (from Stratagene's cDNA Synthesis

[0233] 3. Incubate 37° C., 30 minutes.

[0234] 4. Add 31 μl H₂O and 5 μl 10X STE.

[0235] 5. Size fractionate on a Sephacryl S-500 spin column (poolfractions 1-3).

[0236] 6. Phenol/chloroform extract once.

[0237] 7. Chloroform extract once.

[0238] 8. Add ice cold ethanol to precipitate.

[0239] 9. Place on ice, 10 minutes.

[0240] 10. Spin in microfuge, high speed, 30 minutes.

[0241] 11. Wash with 1 ml 70% ethanol.

[0242] 12. Spin in microfuge, high speed, 10 minutes and dry.

[0243] 13. Resuspend in 10.5 μl TE buffer.

[0244] Do not plate assay. Instead, ligate directly to arms as aboveexcept use 2.5 μl of DNA and no water.

[0245] Package and titer as above.

[0246] Efficiency results:

[0247] gt11: 2.5×10⁶ recombinants with 2.5% background

[0248] ZAP II: 9.6×10⁵ recombinants with 0% background

[0249] Amplification of Libraries (5.0×10⁵ recombinants from eachlibrary)

[0250] 1. Add 3.0 ml host cells (OD₆₆₀=1.0) to two 50 ml conical tube.

[0251] 2. Inoculate with 2.5×10⁵ pfui per conical tube.

[0252] 3. Incubate 37° C., 20 minutes.

[0253] 4. Add top agar to each tube to a final volume of 45 ml.

[0254] 5. Plate the tube across five 150 mm plates.

[0255] 6. Incubate 37° C., 6-8 hours or until plaques are about pin-headin size.

[0256] 7. Overlay with 8-10 ml SM Buffer and place at 4° C. overnight(with gentle rocking if possible).

[0257] Harvest Phage

[0258] 1. Recover phage suspension by pouring the SM buffer off eachplate into a 50-ml conical tube.

[0259] 2. Add 3 ml chloroform, shake vigorously and incubate at roomtemperature, 15 minutes.

[0260] 3. Centrifuge at 2 K rpm, 10 minutes to remove cell debris.

[0261] 4. Pour supernatant into a sterile flask, add 500 μl chloroform.

[0262] 5. Store at 4° C.

[0263] Titer Amplified Library

[0264] 1. Make serial dilutions:

[0265] 10⁻⁵=1 μl amplified phage in 1 ml SM Buffer

[0266] 10⁻⁶=1 μl of the 10⁻³ dilution in 1 ml SM Buffer

[0267] 2. Add 200 μl host (in 10 mM MgSO₄) to two tubes.

[0268] 3. Inoculate one with 10 μl 10⁻⁶ dilution (10⁻⁵).

[0269] 4. Inoculate the other with 1 μl 10⁻⁶ dilution (10⁻⁶).

[0270] 5. Incubate 37° C., 15 minutes.

[0271] 6. Add about 3 ml 48° C. top agar.

[0272] [50 ml stock containing 150 μl IPTG (0.5M) and 375 μl X-GAL (350mg/ml)]

[0273] 7. Plate on 100 mm plates and incubate 37° C., overnight.

[0274] 8. Results:

[0275] gt11: 1.7×10¹¹/ml

[0276] ZAP II: 2.0×10¹⁰/ml

EXAMPLE 2 Enzymatic Activity Assay

[0277] The following is a representative example of a procedure forscreening an expression library prepared in accordance with Example 1.In the following, the chemical characteristic Tiers are as follows:

[0278] Tier 1: Hydrolase

[0279] Tier 2: Amide, Ester and Acetal

[0280] Tier 3: Divisions and subdivisions are based upon the differencesbetween individual substrates which are covalently attached to thefunctionality of Tier 2 undergoing reaction; as well as substratespecificity.

[0281] Tier 4: The two possible enantiomeric products which the protein,e.g. enzyme, may produce from a substrate.

[0282] Although the following example is specifically directed to theabove mentioned tiers, the general procedures for testing for variouschemical characteristics is generally applicable to substrates otherthan those specifically referred to in this Example.

[0283] Screening for Tier 1-hydrolase; Tier 2-amide. Plates of thelibrary prepared as described in Example 1 are used to multiplyinoculate a single plate containing 200 μL of LB Amp/Meth, glycerol ineach well. This step is performed using the High Density ReplicatingTool (HDRT) of the Beckman Biomek with a 1% bleach, water, isopropanol,air-dry sterilization cycle between each inoculation. The single plateis grown for 2h at 37° C. and is then used to inoculate two white96-well Dynatech microtiter daughter plates containing 250 μL of LBAmp/Meth, glycerol in each well. The original single plate is incubatedat 37° C. for 18 h, then stored at −80° C. The two condensed daughterplates are incubated at 37° C. also for 18 h. The condensed daughterplates are then heated at 70° C. for 45 min. to kill the cells andinactivate the host E.coli proteins, e.g. enzymes. A stock solution of 5mg/mL morphourea phenylalanyl-7-amino-4-trifluoromethyl coumarin(MuPheAFC, the ‘substrate’) in DMSO is diluted to 600 μM with 50 mM pH7.5 Hepes buffer containing 0.6 mg/mL of the detergent dodecylmaltoside.

[0284] Fifty μL of the 600 μM MuPheAFC solution is added to each of thewells of the white condensed plates with one 100 μL mix cycle using theBiomek to yield a final concentration of substrate of ˜100 μM. Thefluorescence values are recorded (excitation=400 nm, emission=505 nm) ona plate reading fluorometer immediately after addition of the substrate(t=0). The plate is incubated at 70° C. for 100 min, then allowed tocool to ambient temperature for 15 additional minutes. The fluorescencevalues are recorded again (t=100). The values at t=0 are subtracted fromthe values at t=100 to determine if an active clone is present.

[0285] The data will indicate whether one of the clones in a particularwell is hydrolyzing the substrate. In order to determine the individualclone which carries the activity, the source library plates are thawedand the individual clones are used to singly inoculate a new platecontaining LB Amp/Meth, glycerol. As above, the plate is incubated at37° C. to grow the cells, heated at 70° C. to inactivate the hostproteins, e.g. enzymes, and 50 μL of 600 μM MuPheAFC is added using theBiomek. Additionally three other substrates are tested. They are methylumbelliferone heptanoate, the CBZ-arginine rhodamine derivative, andfluorescein-conjugated casein (˜3.2 mol fluorescein per mol of casein).

[0286] The umbelliferone and rhodamine are added as 600 μM stocksolutions in 50 μL of Hepes buffer. The fluorescein conjugated casein isalso added in 50 μL at a stock concentration of 20 and 200 mg/mL. Afteraddition of the substrates the t=0 fluorescence values are recorded, theplate is incubated at 70° C., and the t=100 min. values are recorded asabove.

[0287] These data indicate which plate the active clone is in, where thearginine rhodamine derivative is also turned over by this activity, butthe lipase substrate, methyl umbelliferone heptanoate, and protein,fluorescein-conjugated casein, do not function as substrates, the Tier 1classification is ‘hydrolase’ and the Tier 2 classification is amidebond. No cross reactivity should be seen with the Tier 2-esterclassification.

[0288] As shown in FIG. 1, a recombinant clone from the library whichhas been characterized in Tier 1 as hydrolase and in Tier 2 as amide maythen be tested in Tier 3 for various specificities. In FIG. 1, thevarious classes of Tier 3 are followed by a parenthetical code whichidentifies the substrates of Table 1 which are used in identifying suchspecificities of Tier 3.

[0289] As shown in FIGS. 2 and 3, a recombinant clone from the librarywhich has been characterized in Tier 1 as hydrolase and in Tier 2 asester may then be tested in Tier 3 for various specificities. In FIGS. 2and 3, the various classes of Tier 3 are followed by a parentheticalcode which identifies the substrates of Tables 3 and 4 which are used inidentifying such specificities of Tier 3. In FIGS. 2 and 3, R₂represents the alcohol portion of the ester and R₁ represents the acidportion of the ester.

[0290] As shown in FIG. 4, a recombinant clone from the library whichhas been characterized in Tier 1 as hydrolase and in Tier 2 as acetalmay then be tested in Tier 3 for various specificities. In FIG. 3, thevarious classes of Tier 3 are followed by a parenthetical code whichidentifies the substrates of Table 5 which are used in identifying suchspecificities of Tier 3.

[0291] Proteins, e.g. enzymes, may be classified in Tier 4 for thechirality of the product(s) produced by the enzyme. For example, chiralamino esters may be determined using at least the following substrates:

[0292] For each substrate which is turned over the enantioselectivityvalue, E, is determined according to the equation below:$E = \frac{\ln\left\lbrack \left( {1 - {c\left( {1 + {ee}_{p}} \right)}} \right\rbrack \right.}{\ln\left\lbrack \left( {1 - {c\left( {1 - {ee}_{p}} \right)}} \right\rbrack \right.}$

[0293] where ee_(p)=the enantiomeric excess (ee) of the hydrolyzedproduct and c=the percent conversion of the reaction. See Wong andWhitesides, Proteins, e.g. enzymes, in Synthetic Organic Chemistry,1994, Elsevier, Tarrytown, N.Y., pp. 9-12.

[0294] The enantiomeric excess is determined by either chiral highperformance liquid chromatography (HPLC) or chiral capillaryelectrophoresis (CE). Assays are performed as follows: two hundred μL ofthe appropriate buffer is added to each well of a 96-well whitemicrotiter plate, followed by 50 μL of partially or completely purifiedprotein, e.g. enzyme, solution; 50 μL of substrate is added and theincrease in fluorescence monitored versus time until 50% of thesubstrate is consumed or the reaction stops, whichever comes first.

EXAMPLE 4 Construction of a Stable, Larde Insert Picoplankton GenomicDNA Library

[0295]FIG. 5 shows an overview of the procedures used to construct anenvironmental library from a mixed picoplankton sample. A stable, largeinsert DNA library representing picoplankton genomic DNA was prepared asfollows.

[0296] Cell collection and preparation of DNA. Agarose plugs containingconcentrated picoplankton cells were prepared from samples collected onan oceanographic cruise from Newport, Oreg. to Honolulu, Hi. Seawater(30 liters) was collected in Niskin bottles, screened through 10 μmNitex, and concentrated by hollow fiber filtration (Amicon DC10) through30,000 MW cutoff polyfulfone filters. The concentrated bacterioplanktoncells were collected on a 0.22 μm, 47 mm Durapore filter, andresuspended in 1 ml of 2X STE buffer (1M NaCl, 0.1M EDTA, 10 mM Tris, pH8.0) to a final density of approximately 1×10¹⁰ cells per ml. The cellsuspension was mixed with one volume of 1% molten Seaplaque LMP agarose(FMC) cooled to 40° C., and then immediately drawn into a 1 ml syringe.The syringe was sealed with parafilm and placed on ice for 10 min. Thecell-containing agarose plug was extruded into 10 ml of Lysis Buffer(1OmM Tris pH 8.0, 50 mM NaCl, 0.1M EDTA, 1% Sarkosyl, 0.2% sodiumdeoxycholate, 1 mg/ml lysozyme) and incubated at 37° C. for one hour.The agarose plug was then transferred to 40 mls of ESP Buffer (1%Sarkosyl, 1 mg/ml proteinase K, in 0.5M EDTA), and incubated at 55° C.for 16 hours. The solution was decanted and replaced with fresh ESPBuffer, and incubated at 55° C. for an additional hour. The agaroseplugs were then placed in 50 mM EDTA and stored at 4° C. shipboard forthe duration of the oceanographic cruise.

[0297] One slice of an agarose plug (72 μl) prepared from a samplecollected off the Oregon coast was dialyzed overnight at 4° C. against 1mL of buffer A (100 mM NaCl, 10 mM Bis Tris Propane-HC1, 100 μg/mlacetylated BSA: pH 7.0 @ 25° C.) in a 2 mL microcentrifuge tube. Thesolution was replaced with 250 μl of fresh buffer A containing 10 mMMgCl₂ and 1 mM DTT and incubated on a rocking platform for 1 hr at roomtemperature. The solution was then changed to 250 μl of the same buffercontaining 4U of Sau3Al (NEB), equilibrated to 37° C. in a water bath,and then incubated on a rocking platform in a 37° C. incubator for 45min. The plug was transferred to a 1.5 ml microcentrifuge tube andincubated at 68° C. for 30 min to inactivate the protein, e.g. enzyme,and to melt the agarose. The agarose was digested and the DNAdephosphorylased using Gelase and HK-phosphatase (Epicentre),respectively, according to the manufacturer's recommendations. Proteinwas removed by gentle phenol/chloroform extraction and the DNA wasethanol precipitated, pelleted, and then washed with 70% ethanol. Thispartially digested DNA was resuspended in sterile H₂O to a concentrationof 2.5 ng/μl for ligation to the pFOS1 vector.

[0298] PCR amplification results from several of the agarose plugs (datanot shown) indicated the presence of significant amounts of archaealDNA. Quantitative hybridization experiments using rRNA extracted fromone sample, collected at 200 m of depth off the Oregon Coast, indicatedthat planktonic archaea in (this assemblage comprised approximately 4.7%of the total picoplankton biomass (this sample corresponds to “PACI”-200m in Table 1 of DeLong et al., high abundance of Archaea in Antarcticmarine picoplankton, Nature, 371:695-698, 1994). Results fromarchaeal-biased rDNA PCR amplification performed on agarose plug lysatesconfirmed the presence of relatively large amounts of archaeal DNA inthis sample. Agarose plugs prepared from this picoplankton sample werechosen for subsequent fosmid library preparation. Each 1 ml agarose plugfrom this site contained approximately 7.5×10⁵ cells, thereforeapproximately 5.4×10⁵ cells were present in the 72 μl slice used in thepreparation of the partially digested DNA.

[0299] Vector arms were prepared from pFOS1 as described (Kim et al.,Stable propagation of casmid sized human DNA inserts in an F factorbased vector, Nucl. Acids Res., 20:10832-10835, 1992). Briefly, theplasmid was completely digested with AstII, dephosphorylated with HKphosphatase, and then digested with BamHI to generate two arms, each ofwhich contained a cos site in the proper orientation for cloning andpackaging ligated DNA between 35-45 kbp. The partially digestedpicoplankton DNA was ligated overnight to the PFOS1 arms in a 15 μlligation reaction containing 25 ng each of vector and insert and 1U ofT4 DNA ligase (Boehringer-Mannheim). The ligated DNA in four microlitersof this reaction was in vitro packaged using the Gigapack XL packagingsystem (Stratagene), the fosmid particles transfected to E. coli strainDH10B (BRL), and the cells spread onto LB_(cm15) plates. The resultantfosmid clones were picked into 96-well microliter dishes containingLB_(cm15) supplemented with 7% glycerol. Recombinant fosmids, eachcontaining ca. 40 kb of picoplankton DNA insert, yielded a library of3.552 fosmid clones, containing approximately 1.4×10⁸ base pairs ofcloned DNA. All of the clones examined contained inserts ranging from 38to 42 kbp. This library was stored frozen at −80° C. for later analysis.

[0300] Numerous modifications and variations of the present inventionare possible in light of the above teachings; therefore, within thescope of the claims, the invention may be practiced other than asparticularly described. TABLE 1 A2 Fluorescein conjugated casein (3.2mol fluorescein/mol casein) CBZ-Ala-AMC t-BOC-Ala-Ala-Asp-AMCsuccinyl-Ala-Gly-Leu-AMC CBZ-Arg-AMC CBZ-Met-AMC morphourea-Phe-AMCt-BOC = t-butoxy carbonyl, CBZ = carbonyl benzyloxy. AMC =7-amino-4-methyl coumarin AA3

AB3

AC3

AD3 Fluorescein conjugated casein t-BOC-Ala-Ala-Asp-AFCCBZ-Ala-Ala-Lys-AFC succinyl-Ala-Ala-Phe-AFC succinyl-Ala-Gly-Leu-AFCAFC = 7-amino-4-trifluoromethyl coumarin.) AE3 Fluorescein conjugatedcasein AF3 t-BOC-Ala-Ala-Asp-AFC CBZ-Asp-AFC AG3 CBZ-Ala-Ala-Lys-AFCCBZ-Arg-AFC AH3 succinyl-Ala-Ala-Phe-AFC CBZ-Phe-AFC CBZ-Trp-AFC AI3succinyl-Ala-Gly-Leu-AFC CBZ-Ala-AFC CBZ-Sewr-AFC

[0301] TABLE 2 L2

LA3

LB3

LC3

LD3

LE3

LF3

LG3

[0302] TABLE 3 LH3

LI3

LJ3

LK3 LL3 LM3

LN3 LO3

[0303] TABLE 4

4-methyl umbelliferone wherein R = G2 β-D-galactose β-D-glucoseβ-D-glucoronide GB3 β-D-cellotrioside β-B-cellobiopyranoside GC3β-D-galactose α-D-galactose GD3 β-D-glucose α-D-glucose GE3β-D-glucuronide GI3 β-D-N,N-diacetylchitobiose GJ3 β-D-fucose α-L-fucoseβ-L-fucose GK3 β-D-mannose α-D-mannose non-Umbelliferyl substrates GA3amylose [polyglucan α1,4 linkages], amylopectin [polyglucan branchingα1,6 linkages] GF3 xylan [poly 1,4-D-xylan] GG3 amylopectin, pullulanGH3 sucrose, fructofuranoside

What is claimed is:
 1. A process of screening clones having DNA from anuncultivated microorganism for a specified small molecule activity,which process comprises: (i) recovering DNA from a DNA populationderived from at least one uncultivated microorganism; and (ii)transforming a host with recovered DNA to produce a library of cloneswhich is screened for the specified small molecule activity.
 2. Theprocess of claim 1 wherein the recovered DNA is amplified.
 3. Theprocess of claim 1 wherein the recovered DNA is ligated into a vector.4. The process of claim 3 wherein the vector into which the recoveredDNA is ligated comprises at least one DNA sequence capable of regulatingproduction of a detectable small molecule activity from said recoveredDNA.
 5. The process of claim 1 wherein the vector into which therecovered DNA has been ligated is used to transform a host.
 6. A processof screening clones having DNA from an uncultivated microorganism for aspecified enzyme activity, which process comprises: screening for aspecified enzyme activity in a library of clones prepared by (i)recovering DNA from a DNA population derived from at least oneuncultivated microorganism; and (ii) transforming a host with recoveredDNA to produce a library of clones which is screened for the specifiedenzyme activity.
 7. The process of claim 6 wherein the recovered DNA isamplified.
 8. The process of claim 6 wherein the recovered DNA isligated into a vector.
 9. The process of claim 8 wherein the vector intowhich the recovered DNA is ligated comprises at least one DNA sequencecapable of regulating production of a detectable enzyme activity fromsaid recovered DNA.
 10. The process of claim 6 wherein the vector intowhich the recovered DNA has been ligated is used to transform a host.